The subject of plant protection against pathogens remains the area of utmost importance in agriculture. Many commercially valuable agricultural crops are prone to infection by plant viruses and fungi capable of inflicting significant damage to a crop in a given season, and drastically reducing its economic value. The reduction in economic value to the farmer in turn results in a higher cost of goods to ultimate purchasers.
Fungal pathogens contribute significantly to the most severe pathogen outbreaks in plants. Plants have developed a natural defense system, including morphological modifications in their cell walls, and synthesis of various anti-pathogenic compounds. See, e.g. Boller, et al., Plant Physiol. 74:442-444 (1984); Bowles, Annu. Rev. Biochem. 59:873-907 (1990); Joosten, et al., Plant Physiol. 89:945-951 (1989); Legrand, et al., Proc. Natl. Acad. Sci. USA 84:6750-6754 (1987); and Roby, et al., Plant Cell 2:999-1007 (1990). Several pathogenesis-related (PR) proteins have been shown to have anti-fungal properties and are induced following pathogen infection. These are different forms of hydrolytic enzymes, such as chitinases and β-1,3-glucanases that inhibit fungal growth in vitro by destroying fungal cell walls. See, e.g. Boller, et al., supra; Grenier, et al., Plant Physiol. 103:1277-123 (1993); Leah, et al., J. Biol. Chem. 266:1464-1573 (1991); Mauch, et al., Plant Physiol. 87:325-333 (1988); and Sela-Buurlage Buurlage, et al., Plant Physiol. 101:857-863 (1993).
Several attempts have been made to enhance the pathogen resistance of plants via recombinant methodologies using genes encoding pathogenesis-related proteins (such as chitinases and β-1,3-glucanases) with distinct lytic activities against fungal cell walls. See, e.g., Broglie, et al., Science 254:1194-1197 (1991); Vierheilig, et al., Mol. Plant-Microbe Interact. 6:261-264 (1993); and Zhu, et al., Bio/Technology 12:807-812 (1994). Recently, two other classes of genes have been shown to have potential in conferring disease resistance in plants. Wu, et al., Plant Cell 7:1357-1368 (1995), reports that a transgenic potato expressing the Aspergillus niger glucose oxidase gene exhibited increased resistance to Erwinia carotovora and Phytophthora infestans. The hypothesis is that the glucose oxidase-catalyzed oxidation of glucose produces hydrogen peroxide, which when accumulates in plant tissues, leads to the accumulation of active oxygen species, which in turn, triggers production of various anti-pathogen and anti-fungal mechanisms such as phytoalexins (see Apostol, et al., Plant Physiol. 90:109-116 (1989) and Degousee, Plant Physiol. 104:945-952 (1994)), pathogenesis-related proteins (Klessig, et al., Plant Mol. Biol. 26:1439-1458 (1994)), strengthening of the plant cell wall, (Brisson, et al., Plant Cell 6:1703-1712 (1994)), induction of systemic acquired resistance by salicylic acid (Chen, et al., Science 162:1883-1886 (1993)), and hypersensitive defense response (Levine, et al., Cell 79:583-593 (1994)).
In addition to the studies on virus resistance in plants, ribosome inactivating proteins (RIPs) have been studied in conjunction with fungal resistance. For example, Logeman, et al., Bio/Technology 10:305-308 (1992), report that an RIP isolated from barley endosperm provided protection against fungal infection to transgenic tobacco plants. The combination of barley endosperm RIP and barley class-II chitinase has provided synergistic enhancement of resistance to Rhizoctonia solani in tobacco, both in vitro and in vivo. See, e.g., Lea, et al., supra; Mauch, et al., supra; Zhu, et al., supra; and Jach, et al., The Plant Journal 8:97-109 (1995). PAP, however, has not shown antifungal activity in vitro. See Chen, et al., Plant Pathol. 40:612-620 (1991), which reports that PAP has no effect on the growth of the fungi Phytophthora infestans, Colletotrichum coccodes, fusarium solani, fusarium sulphureum, Phoma foreata and Rhizoctonia solani in vitro.
Lodge, et al., Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993), report the Agrobacterium tumefaciens-mediated transformation of tobacco with a cDNA encoding wild-type pokeweed antiviral protein (PAP) and the resistance of the transgenic tobacco plants to unrelated viruses. Pokeweed antiviral protein (PAP) is a 29-kDa ribosome inactivating protein that catalytically removes two adenines and a guanine from the sarcin/ricin (S/R) loop of the large rRNA (Endo et al., J. Biol. Chem. 263:8735-8739 (1988); Hudak et al., J. Biol. Chem. 274:3859-3864 (2000) and disrupts binding of elongation factors to the ribosome (Montanaro et al., Biochemical J. 146:127-131 (1975); Osborn et al., European J. of Biocham. 193:401-407 (1990)). Aside from this demonstration of broad spectrum resistance to viruses, it has been demonstrated that when expressed in transgenic plants, PAP also confers broad spectrum antifungal (Zoubenko et al., Nature Biotechnol. 15:922-996 (1997); Zoubenko et al., Plant Mol. Biol. 44:219-229 (2000)) activity. It has also been shown that PAP recognizes its ribosomal substrate by binding to L3 (Hudak et al., J. Biol. Chem. 274:3858-3864 (1999)).
Lodge also reports, however, that the PAP-expressing tobacco plants (i.e., above 10 ng/mg protein) tended to have a stunted, mottled phenotype, and that other transgenic tobacco plants that accumulated the highest levels of PAP were sterile. U.S. Pat. Nos. 5,756,322 and 5,880,322 teach PAP mutants that when produced in plants exhibit less toxicity than wild-type PAP and exhibit biological activities (e.g., resistance to viruses, fungi and other pests) akin to wild-type PAP. It has also been reported that PAP II and PAP II mutants exhibit reduced phytotoxicity compared to wild-type PAP. See Wang, et al., Plant Mol. Biol. 38:957-964 (1998).
The trichothecenes are a family of low molecular weight sesquiterpenoid mycotoxins synthesized by various Fusarium species of fungi. Deoxynivalenol (DON) produced by F. graminearum or F. culmorum that causes fusarium head scab of wheat is a worldwide problem for human health concern and poses a major impact on animal production if present in feeds (Miller et al., Nat. Toxins 5:234-237 (1997)). Other trichothecenes include fusarenon X, trichothecin, verrucarin A, nivalenol, trichodermin, T-2 toxin and diacetoxyscirpenol (DAS). Trichothecenes inhibit peptidyl transferase reaction of protein synthesis by binding to the 60S ribosomal subunit. In addition, they cause membrane damage (Feinberg et al., C. S. 1989. Biochemical mechanism of actions of trichothecene mycotoxins. Pages 27-36 in: Trichothecene mycotoxixosis: Pathophysiological effects, Vol. 1. V. R. Beasley, ed. Boca Raton, Fla., CRC Press. Khachatourians, Canad. J. Physiol. Pharm. 68:1004-1008 (1990); Miller et al., Nat. Toxins 5:234-237 (1997)). Mitterbauer et al., 7th International Congress of Plant Pathology, Edinburgh, Scotland, 5.4.6. (1998) demonstrate that trichothecene resistance in the yeast, Saccharomyces cerevisiae, could result from either alterations in the target of trichothecenes, the ribosomal protein L3 or the increased drug efflux due to over-expression of a membrane transporter protein encoded by the PDR5 gene.
L3 is a highly conserved ribosomal protein that participates in the formation of the peptidyltransferase center that in turn allows elongation of the ribosome along the messenger RNA (mRNA). Hampl, et al., J. Biol. Chem. 256:2284-2288 (1981); Noller, J. Bacteriol. 175:5297-5300 (1993). L3 also plays an essential role in the catalysis of peptide bond formation. See, Green, et al., Annu. Rev. Biochem. 66:679-716 (1997). This is an essential step in protein synthesis in yeast, animals and higher plants. L3 is encoded by the rpl3 gene. Trichodermin, a substituted 12,13-epoxytrichothecene, is known to inhibit peptide bond formation by binding to the peptidyl transferase center. A mutation in the Rpl3 gene, designated tcm-1, which contains a single amino acid substitution of tryptophan to cysteine at position 255 (i.e., W255C) was initially identified in yeast by conferring resistance to trichodermin (Fried, et al., Proc. Natl. Acad. Sci. USA 78:238-242 (1981)). U.S. Pat. No. 6,060,646 to Harris, et al., teaches modified peptidyl transferase (L3) genes that provide resistance to trichothecene mycotoxins, such as the tcm-1 gene. Transgenic plants transformed with genes encoding L3 proteins are disclosed in WO 00/39291. The L3 proteins include wild-type L3, spontaneously occurring mutants and other non-naturally occurring mutants. It also teaches plants transformed with L3 genes and genes encoding ribosome inactivating proteins such as PAP.
Studies by Muhitch et al., Plant Science 157:201-207 (2000) demonstrated that tobacco plants transformed with either the Saccharomyces cerevisiae gene PDR5, which encodes a multi-drug transporter, or with the Fusarium sporotrichioides gene TRI101, which encodes a trichothecene 3-O-acetyltransferase, showed increased tolerance to the trichothecene 4,15-diacetoxyscirpenol (DAS). Even more recently, Harris et al., Physiol. Mol. Plant. Path. 58:173-181 (2001), showed that transgenic tobacco tissues transformed with a modified Rpl3 gene from rice displayed resistance to DON.